Jan A. Ruzicka

Interpreting the Conductance Blockades of DNA Translocations through Solid-State Nanopores

Solid-state nanopore electrical signatures can be convoluted and are thus challenging to interpret. In order to better understand the origin of these conductance changes, we investigate the translocation of DNA through small, thin pores over a range of voltage. We observe multiple, discrete populations of conductance blockades that vary with applied voltage. To describe our observations, we develop a simple model that is applicable to solid-state nanopores generally. These results represent an important step toward understanding the dynamics of the electrokinetic translocation process.

Selective Detection and Quantification of Modified DNA with Solid-State Nanopores

We demonstrate a solid-state nanopore assay for the unambiguous discrimination and quantification of modified DNA. Individual streptavidin proteins are employed as high-affinity tags for DNA containing a single biotin moiety. We establish that the rate of translocation events corresponds directly to relative concentration of protein-DNA complexes and use the selectivity of our approach to quantify modified oligonucleotides from among a background of unmodified DNA in solution.

Nutrition, HIV, and drug abuse: the molecular basis of a unique role for selenium.

&NA;HIV‐infected injection drug users (IDUs) often suffer from serious nutritional deficiencies. This is a concern because plasma levels of micronutrients such as vitamin B12, zinc, and selenium have been correlated with mortality risk in HIV‐positive populations. Injection drug use also increases lipid peroxidation and other indicators of oxidative stress, which, combined with antioxidant deficiencies, can stimulate HIV‐1 replication through activation of NF‐&kgr;B transcription factors, while weakening immune defenses. As detailed herein, these prooxidant stimuli can also increase the pathogenic effects of HIV‐1 by another mechanism, involving viral selenoproteins. Overlapping the envelope coding region, HIV‐1 encodes a truncated glutathione peroxidase (GPx) gene (see #6 in reference list). Sequence analysis and molecular modeling show that this viral GPx (vGPx) module has highly significant structural similarity to known mammalian GPx, with conservation of the catalytic triad of selenocysteine (Sec), glutamine, and tryptophan. In addition to other functions, HIV‐1 vGPx may serve as a negative regulator of proviral transcription, by acting as an NF‐&kgr;B inhibitor (a known property of cellular GPx). Another potential selenoprotein coding function of HIV‐1 is associated with the 3′ end of the nef gene, which terminates in a conserved UGA (potential Sec) codon in the context of a sequence (Cys‐Sec) identical to the C‐terminal redox center of thioredoxin reductase, another cellular regulator of NF‐&kgr;B. Thus, in combination with known cellular mechanisms involving Se, viral selenoproteins may represent a unique mechanism by which HIV‐1 monitors and exploits an essential micronutrient to optimize its replication relative to the host.

Sequence-Specific Recognition of MicroRNAs and Other Short Nucleic Acids with Solid-State Nanopores

The detection and quantification of short nucleic acid sequences has many potential applications in studying biological processes, monitoring disease initiation and progression, and evaluating environmental systems, but is challenging by nature. We present here an assay based on the solid-state nanopore platform for the identification of specific sequences in solution. We demonstrate that hybridization of a target nucleic acid with a synthetic probe molecule enables discrimination between duplex and single-stranded molecules with high efficacy. Our approach requires limited preparation of samples and yields an unambiguous translocation event rate enhancement that can be used to determine the presence and abundance of a single sequence within a background of nontarget oligonucleotides.

Nanopore Analysis of Single-Stranded Binding Protein Interactions with DNA

We study the binding of E. coli single-stranded binding protein (SSB) to single-stranded DNA (ssDNA) using a solid-state nanopore assay. We find that saturated nucleoprotein complexes can be distinguished easily from free SSB, ssDNA, or double-stranded DNA individually and demonstrate that the high affinity of SSB for ssDNA can be exploited to achieve high-fidelity differentiation from duplex molecules in a mixture. We then study nucleoprotein filament formation by systematically varying the amount of SSB relative to ssDNA. We observe a concomitant shift in the mean amplitude of electrical events that is consistent with weakly cooperative binding. Finally, we compare circular and linearized ssDNA saturated with SSB and use the results to infer structural details of the nucleoprotein complex.

Detecting DNA Depurination with Solid-State Nanopores

Among the different types of DNA damage that occur endogenously in the cell, depurination is especially prevalent. These lesions can initiate mutagenesis and have been implicated in a variety of diseases, including cancer. Here, we demonstrate a new approach for the detection of depurination at the single-molecule scale using solid-state nanopores. We induce depurination in short duplex DNA using acidic conditions and observe that the presence of apurinic sites results in significantly slower dynamics during electrokinetic translocation. This procedure may be valuable as a diagnostic for in situ quantification of DNA depurination.

Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry

Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3′ end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation.